Glyceraldehyde 3-phosphate is an essential enzyme in carbohydrate metabolism in all cells. The mammalian enzyme exhibits negative cooperativity in ligand binding and half-of-sites reactivity with some reagents. We plan to examine three aspects of its structure, function and regulation. 1. Interaction of coenzymes with various forms of the enzyme. In this work we will determine how coenzyme binding is affected by the presence of acyl or alkyl substituents at the active sites. These forms of the enzyme are models for the normal intermediates in the kinetic mechanism and should represent the enzyme species to which NAD and NADH normally bind. Part of the work will be directed at finding out, through binding studies and kinetic measurements, how ligands or substituents on one subunit affect the behavior of adjacent subunits to produce negative cooperativity, half-of-sites reactivity and other phenomena related to cross-subunit interactions. 2. Interaction of inorganic phosphate with the enzyme. Binding studies using ultrafiltration and equilibrium dialysis as well as kinetic studies will be used to determine how phosphate, one of the substrates for the enzyme, interacts with the active site. We plan to find out if there is negative cooperativity in its binding and whether other ligands affect its interaction with the enzyme. 3. Naturally occurring inhibitors of glyceraldehyde 3-phosphate dehydrogenase. Several preliminary reports have shown that there are inhibitors of the enzyme in blood and urine. We want to isolate and identify these inhibitors and characterize their effect on the enzyme as determined by their inhibition of enzyme activity.